In the raising of animals for commercial purposes, various strains of pneumonia causing organisms can be a significant cause of animal death. More particularly in the raising of cattle, "Shipping Fever" pneumonia is the major cause of sickness and mortality in feedlot cattle in North America. Although several respiratory viruses and bacteria have been implicated in the pathogenesis of the syndrome, the principal well known organism isolated is Pasteurella haemolytica serotype A1. The disease can be reproduced experimentally by intratrachael inoculation of the microorganism. Bacterins incorporating P. haemolytica have been in use for more than sixty years in preventing this disease without significant impact on disease control. Evidence from field studies and experimental trials suggests an adverse effect of vaccination using the bacterins. Animals vaccinated with inactivated whole cell bacterins frequently show a higher incidence of pneumonia and more severe lesions at post mortem than do unvaccinated animals. This occurs despite the induction of serum antibody to P. haemolytica cell surface antigens, measured by bacterial agglutination or passive hemaglutination techniques. This has resulted in considerable confusion with respect to how this type of pneumonia can be prevented. Paradoxically, the occurrence of an analogous response as a result of natural or experimental infection with live bacteria has resulted in developing a degree of immunity to pneumonia in so infected animals.
It has been determined that culture supernatant of Pasteurella haemolytica is cytotoxic to bovine but not porcine cells, as reported in "Cytotoxin of Pasteurella haemolytica Acting on Bovine Leukocytes", P. E. Shewen and B. N. Wilkie, Infection and Immunity, January 1982, Vol. 32 No. 1, 91. Work was then directed to the production of the cytotoxin by culture of Pasteurella haemolytica and the conversion of culture supernatant into a vaccine. The impetus for development of such a vaccine partially resulted from the generation of anti-toxic immune response after natural exposure of animals to P. haemolytica. Calves vaccinated with leukotoxic culture supernate isolated from the culture of P. haemolytica produced both anti-toxic and bacterial agglutinating antibody. The so vaccinated calves were more resistant to experimental challenge than were counterparts vaccinated with bacterins or unvaccinated calves, as reported in "Immunity to Pasteurella haemolytica Cytotoxin", P. E. Shewen and B. N. Wilkie, 1982, Conf. Res. Workers Animal Disease, Chicago, Ill., Abstract 138.
As a result, production in vitro of the cytotoxin by Pasteurella haemolytica has become very much of interest in an attempt to make a suitable vaccine on a commercial basis for counteracting "Shipping Fever" pneumonia. To date, the only viable technique for the in vitro production of cytotoxin has required the addition of serum or blood to the culture medium and in particular the use of fetal calf serum. Any attempt to manufacture the cytotoxin in a serum-free medium by culturing P. haemolytica has resulted in what was thought to be an absence of produced cytotoxin because any assay for the cytotoxin was negative. Fetal calf serum is used as a seven percent solution which has been established to be the minimum amount needed to permit production of toxic culture supernate in RPMI 1640 medium. With the use of fetal calf serum or other stabilizing serum, heat-labile leukotoxin is made by culturing the P. haemolytica and harvesting the cytotoxic supernatant after approximately one hour of growth at 37.degree. C. in the manner reported in the aforementioned article "Cytotoxin of Pasteurella haemolytica Acting on Bovine Leukocytes". The use of serum and particularly fetal calf serum in the manufacture of the cytotoxin complicates analysis of P. haemolytica antigens present in culture supernate, greatly increases the cost for vaccine production and introduces potentially harmful extraneous antigens into the vaccine preparations. Furthermore, the presence of the serum in the supernate maintains activity of the toxin, which is undesirable in the vaccine preparation.